The present invention relates to an antibody which is novel and useful in that it has specific affinity for PACAP, and more particularly to an antibody useful for development of assays of PACAP on the basis of antigen-antibody reactions or for diagnosis and treatment of diseases related to PACAP.
Various hormones secreted by brain hypothalami and hypophyses have been known. Examples thereof include thyrotropin releasing hormone, luteinizing hormone releasing hormone, somatostatin, adrenocorticotropic hormone, growth hormone and prolactin. Action thereof has been studied in detail. Recently, a novel bioactive substance of hypothalamic origin other than these hormones was studied based upon adenylate cyclase activity, and consequently a peptide consisting of 38 amino acid residues which had not been reported till then was discovered from sheep hypothalami. This peptide was named "PACAP38NH.sub.2 " and has a structure represented by the following formula (SEQ ID NO:2): EQU His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu Gly Lys Arg Tyr Lys Gln Arg Val Lys Asn Lys-NH.sub.2
It was disclosed in applications for patents (Japanese Patent Application Nos. 1-155791/1990 and 1-284771/1990) on cDNA of sheep PACAP38, and an application for a patent (Japanese Patent Application No. 1-259924/1990) on the partial structure of cDNA of human PACAP38 that the amino acid sequence of the mature portion of sheep PACAP38 was the same as that of human PACAP38, and that some amino acids of the precursors thereof were substituted. It is deduced from the position of continuous basic amino acids shown in the cDNA sequence of PACAP38NH.sub.2 that PACAP27NH.sub.2, in addition to PACAP38NH.sub.2, will exist as a peptide cut out of the precursor.
In fact, according to subsequent studies PACAP27NH.sub.2 was also isolated from sheep hypothalami, in addition to PACAP38NH.sub.2. The structure thereof is represented by the following formula (SEQ ID NO:2):
PACAP27NH.sub.2 EQU His Ser Asp Gly Ile Phe Thr Asp Ser Tyr Ser Arg Tyr Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Ala Ala Val Leu-NH.sub.2
PACAP38NH.sub.2 and PACAP27NH.sub.2 are hereinafter represented by the general term of "PACAP". The 28 amino acid residues on the N-terminal side of PACAP38NH.sub.2 containing PACAP27NH.sub.2 show 68% homology with vasoactive intestinal polypeptide (VIP) well known as a brain-gut peptide. However, it has been reported that the adenylate cyclase activating activity of PACAP exceeds at least 1,000 times that of VIP.
Thus, the action of PACAP is anticipated to be different from that of VIP, and a deep interest is taken in the physiological role thereof and the relation thereof to the pathology.
Although the interest in PACAP is increased as described above, basic physiological information such as existing sites other than hypothalami of PACAP and a plasma level thereof is scarcely obtained, and the relation thereof to the pathology is also unknown. This is mainly caused by that any monoclonal antibodies specifically recognizing PACAP have hitherto not been prepared and that any immunoassays for assaying PACAP specifically and highly sensitively have not been developed. These immunological procedures are considered to be one of the most effective means to study PACAP, particularly the metabolic pathways thereof, the secretory mechanism thereof, the receptor system thereof, the relation thereof to the pathology and the like collectively. The establishment of these procedures has therefore been earnestly desired in various fields.
Previously, competitive radioimmunoassays (RIA) generally using one kind of antibody and enzyme immunoassays (EIA) have been developed and employed to assay low molecular weight peptides such as PACAP. On the other hand, sandwich immunoassays using two kinds of antibodies have the advantages of (1) improving the specificity of assay systems because of the use of two kinds of antibodies and (2) being little affected by nonspecific interfering factors because of the use of the antibodies in large excess to substances to be assayed. Until now, however, it has been unknown at all whether or not the low molecular weight peptides having no disulfide linkage such as PACAP can be assayed with high sensitivity by the sandwich methods. Namely, in case of the low molecular weight peptides having no disulfide linkage such as PACAP, the possibility is conceivable that binding sites of two kinds of antibodies are in so close proximity to each other as to exert influences such as steric hindrance, which results in difficulty of the establishment of highly sensitive sandwich methods.